Obesity is at epidemic proportions in the United States and it contributes to the development of life-threatening illnesses including heart disease and type 2 diabetes. Recent studies have demonstrated a significant degree of adipocyte turnover in adult human adipose tissue, indicative of ongoing production of mature adipocytes from precursor preadipocytes. Therapies that target molecular mechanisms of adipogenesis have the potential to be effective in the treatment and prevention of obesity. The development of adipocytes from precursors present in adipose tissue is not well understood and research in this area has been limited by our inability to precisely identify this cell type. Moreover, the unique cohort of genes that define a cell as a functional preadipocyte has not been determined. As such, there remains a pressing need to develop new molecular tools to study the fat cell precursor and to identify and characterize novel pathways and molecular programs of adipogenesis. Studies with preadipocyte-expressed inhibitors of adipogenesis conducted to date indicate that this gene cohort may be a particularly rich source of regulators of the adipogenic process. This research project is designed to address gaps in our knowledge of the adipocyte lineage and preadipocyte/adipocyte biology via comprehensive approaches to identify and study genes that define functional preadipocytes. To address this we have a developed and validated a new white preadipocyte cell culture model (ScAP-23) derived from murine white adipose tissue. We will utilize this model and the highly characterized 3T3-L1 white preadipocyte model and other cell lines and analysis of preadipocyte-containing populations of WAT. Our approaches will integrate transcriptional profiling data analyzed with bioinformatics-based methodologies and functional testing of identified candidate genes and molecular pathways in preadipocytes and adipocytes. In Aim 1 we will identify novel preadipocyte-expressed inhibitors of adipogenesis and the mature adipocyte phenotype using gain of function studies conducted in preadipocytes in culture and with in vivo transplantation. In Aim 2 we will conduct genome-wide transcriptional analysis to identify genes highly enriched and selectively expressed in committed preadipocytes. Candidate preadipocyte commitment/competence genes will be tested for roles in commitment of mesenchymal precursors to the preadipocyte lineage by gain of function studies. The effect of their knockdown on preadipocyte commitment and differentiation will also be determined. We predict these studies will uncover new molecular pathways for the adipocyte lineage. In addition, our work will generate new gene expression datasets highly useful to the research community and will form an important foundation for the development of new molecular tools to study preadipocytes. PUBLIC HEALTH RELEVANCE: By determining the molecular processes underlying the formation and function of adipose tissue we can better define therapies and treatments for obesity. While there is considerable information on the genes which function in adipocytes, those which that are important for the cell type from which mature adipocytes arise, termed preadipocytes, remain largely unexplored. By better defining these genes and their functions we aim to uncover unique ways to study the development of adipose tissue and control the process of conversion of preadipocytes to adipocytes that will ultimately lead to better understanding and treatment of obesity.